Identifizierung des MYBL2-vermittelten Tumorsuppressor Signalweges und seiner wichtigsten kooperierenden Mutationen in myeloischen Erkrankungen
Zusammenfassung der Projektergebnisse
Almost all patients with Myelodysplastic Syndromes (MDS) present with anemia as a result of disturbed erythropoiesis. Previously, we identified the transcription factor MYBL2 as one of the most significantly downregulated genes in CD34+ cells from MDS patients with a normal karyotype. This finding was surprising as MYBL2 is a ubiquitously expressed transcription factor that is required for cell cycle progression and its complete inactivation results in growth arrest and cell death. Indeed, we found that MYBL2 genetically behaves as a gene dosage-dependent tumor suppressor, i.e. its downregulation confers a competitive advantage to hematopoietic progenitor cells in the context of MDS. In this project we aimed to uncover the basis of this tumor suppressing activity. To this end, we first developed a doxycycline-dependent model based on primary cells. We identified the knockout of the transcriptional co-repressor Bcor, which is frequently mutated in MDS, as collaborating event in combination with downregulation of Mybl2 to immortalize primary hematopoietic stem and progenitor cells (HSPCs) in vitro. We used CRISPR/Cas9 genome editing to knockout Bcor and inducible RNA interference (RNAi) to downregulate Mybl2. Immortalized cells showed an immature immunophenotype marked by lack of lineage markers and, functionally, an impaired ability to differentiate towards mature erythroid cells. Differential gene expression after restoration of Mybl2 expression granted a first insight into potentially relevant target genes such as Cd34 or Erg. However, an additional experiment using a later sampling time point after Mybl2 restoration is underway. In support of the vitro model, lethally irradiated mice transplanted with such genetically modified Mybl2 knockdown/Bcor knockout cells showed an inferior survival associated with an expansion of these genetically modified cells in the bone marrow. Overall, the mice suffered from disturbed hematopoiesis. Yet, also in vivo, erythropoiesis was particularly affected. Thus, our Bcor knockout/Mybl2 knockdown model revealed a critical role for Mybl2 particularly in erythroid differentiation. In agreement, the reanalysis of external single cell sequencing data revealed that high levels of Mybl2 are required along the erythroid differentiation trajectory. In conclusion, we undertook the challenging task to establish immortalized cell lines that would allow to uncover the Mybl2-associated phenotype which is apparently mainly affecting erythroid differentiation. While we generated immortalized cell lines using three different RNAi targeting sequences, selecting subpopulations and the time point for analysis was a challenge that we have not yet fully solved.
Projektbezogene Publikationen (Auswahl)
-
Gene dosage insufficiency of Mybl2 in concert with knockout of an epigenetic modifier confers a competitive advantage to hematopoietic stem and progenitor cells. 4th Essen Translational Oncology Symposium (2021)
Wossidlo, N., Langer, K.S., Horn, P.A., Heinrichs S.
-
In vitro and in vivo modelling of gene-dosage insufficiency of Mybl2 in Myelodysplastic Syndromes. 10th International Meeting of the Stem Cell Network (2021)
Wossidlo, N., Langer, K.S., Horn, P.A., Heinrichs S.
